ICANCERRESEARCH57, 3640-3643. September I. 19971 Advances in Brief Role for E2F in DNA Damage-induced Entry of Cells into S Phase1 Yinyin Huang, Takatoshi Ishiko, Shuji Nakada, Taiju Utsugisawa, Tomohisa Kato, and Zhi-Min Yuan2 Division of Cancer Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 Abstract Materials and Methods Mammalian cells respond to ionizing radiation (IR) with transient cell Cell Culture and Cell Cycle Analysis. HumanHL-60cells weregrownin cycle arrest and induction of apoptosis. Here we show that IR increases RPM! 1640 containing 10% heat-inactivated fetal bovine scram, 1 mM L the expression of the E2F-1 transcription factor and the entry of cells into glutamine, 100 units/mI penicillin, and 100 @zg/mlstreptomycin. The null S phase. E2F-1 transactivation function is inhibited by cydlinA-kinaseto pCMV-HA vector and pCMV-HA-DP-l'27410 deletion mutant were intro ensure orderly progression through S phase. However, in contrast to duced into HL-60 cells by electroporation. Stable lines were established by proliferating cells, IR treatment results in down-regulation of cydin A- selection in G418. Irradiation was performed at room temperature in a Gam kinase. Expression of a dominant negative form of the E2F heterodimeric macell 1000 (Atomic Energy of Canada, Ottawa, Canada) with a ‘37Cssource partner DP-1 confirmed the involvement of E2F in IR-induced S-phase emitting a fixed dose of 13.3 Gy/min. DNA content was assessed by staining entry. These fmdings also support opposing signals involving the induc ethanol-fixed cells with propidium iodide and monitoring by FACScan. Cell tion of E2F and the down-regulation ofcydin A-kinase in the IR response. cycle distribution was determined with a ModFit LT program (Verity Software House, Inc.). Cells were labeled with [3H]thymidine (DuPont New England Nuclear) for 4 h after exposure to IR. Introduction Immunoblot Analysis. Cells were lysed in 1% NP4Olysis buffer. Proteins The cellular response to W@ includes transient cell cycle arrest, were resolved by SDS-PAGE, transferred to nitrocellulose filters, and analyzed with anti-E2F-1 (SC-25l; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), activation of DNA repair, and, in the event of irreparable damage, anti-E2F-2 (SC-633), anti-E2F-3, anti-E2F-4 (SC-866), and anti-PCNA (SC induction of apoptosis. Cells also respond to IR with the induction of 56). Proteins were detected with an enhanced chemiluminescence system early response genes (1—4).The findings that IR induces c-jun gene (Amersham, Arlington Heights, IL). expression (1—3)and that Jun/AP-l is involved in GdGI to S-phase Immunoprecipitates and Kinase Assays. Cell lysates were incubated progression (5, 6) provided support for an IR response involving entry with anti-cyclin A (SC-239) for 2 h at 4°C and then for 60 mm with protein into S phase. IR also induces transcription of the EGR-l early re A-Sepharose. Immunoprecipitates were analyzed by histone Hi kinase assays sponse gene that encodes a protein implicated in mitogenesis (2, 4, 7). (23) or immunoblotting with anti-cyclin A, anti-Cdk2 (SC-l63), or anti-Cdc2 (SC-54). Indeed, other studies have shown that after a transient delay in G@, DNA Fragmentation Analysis. DNA was isolated as describedand ana irradiated cells exhibit a reduction in the percentage of G@cells that is lyzed in 2% agarose gels (10). temporally and functionally associated with the activation of early response genes (8, 9). Moreover, this entry into S phase is temporally Results and Discussion related to the induction of apoptosis in certain cells (10). Flow cytometry was performed on JR-treated cells to assess the The E2F family of transcription factors (E2F-1 through E2F-5, relationship between cell cycle distribution and the induction of DP-l , and DP-2) regulates the expression of genes whose products apoptosis. Exposure to 10 Gy of JR resulted in the time-dependent control the entry into and execution of S phase (1 1). The transcrip appearance of cells with sub-G1 DNA content (Fig. 1A) and internu tional activity of E2F-l is repressed by the dephosphorylated form of cleosomal DNA fragmentation (data not shown). IR exposure was Rb (12). Phosphorylation of Rb by cyclin-dependent kinases before also associated with an increase in S-phase cells that corresponded entry into S phase disrupts the interaction between Rb and E2F-1 (12) temporally with an induction of sub-G1 DNA populations (Fig. lA). and renders E2F-1 capable of activating gene expression (12). The The accumulation of cells with both increased sub-G1 and S-phase E2F proteins form active complexes as heterodimers with DP-1 or DNA content was also dose dependent (Fig. lB). An increase in DP-2 (13—15).DP-l mutants that retain E2F binding but not DNA S-phase cells may be the result of S-phase arrest or increased DNA binding arrest cells in G1 (16). Moreover, overexpression of E2F synthesis. Studies of [3H]thymidine incorporation demonstrated that induces entry into S phase and DNA synthesis (17—20).Overexpres the IR-induced accumulation of S-phase cells is due to an increase in sion of E2F-l also leads to apoptosis by p53-dependent mechanisms DNA synthesis (Fig. lC). These findings suggested that the IR-treated (18, 19, 21). The finding that E2F-1@ mice exhibit defects in cells exhibit increased entry into S phase and induction of apoptosis. apoptosis and aberrant cell proliferation has suggested that E2F-l Whereas overexpression of E2F-l induces entry into S phase (17— functions to regulate apoptosis and suppress growth (22). 20), we asked whether IR affects E2F-1 levels. The results demon The present studies demonstrate that the cellular response to IR strate an increase in E2F-l protein that was detectable at 3 h after involves two conflicting signals, induction of E2F-l and down-regu exposure to 10 Gy and more apparent at 6—24h (Fig. 2A). Other lation of cyclin A-kinase activity. studies have demonstrated that overexpression of E2F-4 can drive cell cycle progression (24). However, IR had no detectable effect on Received 6/11/97; accepted 7/14/97. E2F-4 levels (Fig. 2B). E2F-2 and E2F-3 were undetectable in control The costs of publication of this article were defrayed in part by the payment of page and irradiated cells (data not shown). As observed for entry into S charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. phase, the induction of E2F-l was dose dependent (Fig. 2B). To define I SupportedbyUSPHSGrantCA55241awardedbytheNationalCancerInstitute, in part the basis for IR-induced E2F-l levels, we assessed the effects Department of Health and Human Services. of IR on E2F-l mRNA levels and E2F-l protein stability. Northern 2To whom requests for reprints should be addressed. Phone: (617) 632-3142; Fax: (617) 632-2934. analysis (1) demonstrated IR-induced increases in E2F-l transcripts, 3 The abbreviations used are: IR, ionizing radiation; Rb, retinoblastoma protein. whereas there was no effect on actin mRNA levels (data not shown). 3640 Downloaded from cancerres.aacrjournals.org on April 28, 2015. © 1997 American Association for Cancer Research. ROLE FOR E2F IN SR-INDUCEDS-PHASE ENTRY A. .@ E z B. DNAContent .@ Coa@ 250 500 750 1000 1500 El I I Fig. 1. W-induced apoptosis correlates with the entry of cells into S phase. A, @I I I HL-60 cells were exposed to 10 Gy of IR. At the indicated times, cells were zil fixed with 70% ethanol, stained with propidiurn iodide, and subjected to fluorescence-activated cell-sorting analysis. B, cell cycle analysis was per formed 7 h after exposure to the indicated doses of IR (cGy). C, cells were DNA Content exposed to the indicated ER doses. After 3 h, the cells were incubated with 1 pCi/well [3Hjthymidine for 4 h at 37°C.Thecells were harvested, washed, and C. monitored for tritium incorporation. 120 C? @80 @60 40 0 10 20 IR(Gy) A. IR — IR b@ I 0 C .@ .C .C .= .C .C .@ .C @_l I @@@ q @-.cv,Coacsa ‘@. C -= .@ I.) 2 (V)co IC)0 ‘J -.*e.... -‘-E2F-1 ,l_.1..@ HistoneHi @@@ —5_S•@ cyclinA B. 01 cGy c@ ‘@C@JtoN-o to @ — Cdc2 .- __._• -@--E2F-1 .@__@ø— E2F-4 Cdk2 Fig. 3. IR induces down-regulation of cyclin A-kinase activity. Lysates from control and IR (10 Gy)-treated cells were subjected to immunoprecipitation with anti-cyclin A. PCNA TheprecipitateswereassayedforhistoneHl phosphorylation(upperpanet)andsubjected to immunoblotting with anti-cyclin A, anti-Cdk2, or anti-Cdc2. Fig. 2. Cellular E2F-1 levels are induced by IR in a time- and dose-dependent manner. C. Control IR A, cell lysates prepared at the indicated times after exposure to 10 Gy of IR were subjected to immunoblot analysis with anti-E2F-l and anti-PCNA. B, lysates prepared from cells ac .@ .c .c .c .c harvested 7 h after exposure to the indicated doses of lR were subjected to immunoblot o,-csJcv)@tO o'-eJc0)'@.tt) analysis with anti-E2F-1, anti-E2F-4, and anti-PCNA. C, control and IR (10 Gy)-treated cells were pulse-labeled for 30 mm with 100 @.tCilml[35S]methionine-cysteinemix. The cells were chased in medium supplemented with a 10-fold excess of cold methionine for !@-@ @-E2F-1 the indicated times. Lysates were subjected to immunoprecipitation with anti-E2F-l. The precipitates were monitored by SDS-PAGE and autoradiography. No significant differ ence in half-life of the E2F-l protein was observed after IR treatment in three separate experiments.